Summary

The SGK1 protein belongs to the AGC gene family of kinases that are
regulated by phosphorylation mediated by PDK1. SGK1 regulation is accomplished
by several pathways including growth-factor and stress-mediated signaling. We
have expanded the analysis of SGK1 regulation in epithelial cells. We used
HA-tagged SGK1 to transiently transfect MDCK cells and study the regulation of
SGK1 upon stimulation with HGF, cAMP or upon adhesion of the cells to
immobilized fibronectin. In addition, we studied the regulation of SGK1
activity by small GTP-binding proteins of the Rho family.

Treatment of MDCK cells with HGF leads to a time-dependent activation of
SGK1 that is blocked by wortmanin. This activation requires the conserved
phosphorylation site present in the activation loop of the kinase (T256 in
SGK1) and the phosphorylation site present in a hydrophobic domain at its
C-terminus (S422 in SGK1), which are targets for PDK1/PDK2-mediated regulation
of SGK1. We tested whether SGK1 could be activated by cAMP as it contains a
putative PKA site. We were unable to demonstrate a significant activation of
HA-SGK1 by cAMP stimulation under conditions where we detect cAMP-mediated
phosphorylation of the transcription factor CREB.

Cotransfection of SGK1 with activated small GTP-binding proteins revealed
that Rac1, but not Rho or Rap1, induces activation of SGK1. However, this
activation was wortmanin insensitive and dominant-negative Rac1 did not
inhibit the HGF-mediated activation of SGK1. Adhesion of MDCK cells to
immobilized fibronectin also leads to activation of SGK1. However, it appears
that the integrin-mediated activation of HA-SGK1 differs from AKT activation
in the fact that AKT phosphorylation was blocked by wortmanin (or LY294002)
whereas HA-SGK1 was not. The adhesion-dependent activation, however, requires
the intact phosphorylation sites of SGK1. Co-transfection of HA-SGK1 with
RacV12 results in increased activity in adherent cells compared with HA-SGK1
alone. Since RacN17 failed to inhibit adhesion dependent-activation of SGK1,
it suggests that integrin activation is achieved by a parallel Rac-independent
pathway.

The activation of SGK1 by HGF and integrin provides a link between
HGF-mediated protection of MDCK from de-attachment induced apoptosis
(anoikis). We demonstrate that dephosphorylation of the transcription factor
FKRHL1 induced by cell de-attachment is prevented by activated SGK1,
suggesting that SGK1 regulates cell survival pathways.

In summary, we demonstrate that SGK1 activation could be achieved through
signaling pathways involved in the regulation of cell survival, cell-cell and
cell-matrix interactions. SGK1 activation can be accomplished via HGF,
PI-3K-dependent pathways and by integrin-mediated, PI-3K independent pathways.
In addition, activation of SGK1 by the small GTP-binding protein Rac1 has been
observed.

Although it is well established that the phosphorylation of SGK at T256 in
the activation loop is carried out by PDK1, the kinase(s) involved in the
phosphorylation of the site at S422 is still poorly defined. The regulation of
AKT via the PDK2 site has been described as accomplished either by a
`modified' PDK1 (
Balendran et al.,
1999), an autophosphorylation event
(
Toker and Newton, 2000), or
directly (
Delcommenne et al.,
1998) or indirectly (
Lynch et
al., 1999) by the integrin-linked kinase, ILK. It remains to be
determined whether a similar process regulates SGK activity. However, it is
known that phosphorylation of the S422 is required for maximal activation of
SGK by enhancing phosphorylation of the activation loop residue T256
(
Kobayashi and Cohen, 1999;
Kobayashi et al., 1999;
Park et al., 1999). Regulation
of SGK activity by extracellular stimuli, including insulin, insulin-like
growth factor, serum or oxidative stress
(
Kobayashi and Cohen, 1999;
Park et al., 1999) has been
described. On the basis of these studies carried out by Kobayashi and Cohen
(
Kobayashi and Cohen, 1999), a
model for the activation of SGK has been proposed that involves (1)
IGF-1-induced production of PIP3 that activates a PDK2-like enzyme leading to
phosphorylation of S422 and (2) PDK1 phosphorylation of T256. This model is an
analog of the proposed regulation of p70S6k
(
Alessi et al., 1998;
Pullen et al., 1998) and
represents an example of a phosphorylation-dependent substrate for PDK1
(
Vanhaesebroeck and Alessi,
2000). Since SGK activity has been described as being regulated by
extracellular stimulation that leads to PI-3K activation, we have investigated
whether SGK activation could be achieved through signaling pathways involved
in the regulation of cell-cell and cell-matrix interactions. We demonstrate
that in the epithelial cell line MDCK, SGK activation can be accomplished via
a scatter-factor-mediated PI-3K-dependent pathway and by an integrin-mediated
PI-3K independent pathway. In addition, activation of SGK by the small
GTP-binding protein Rac1 has been observed.

Cell culture, transfection and stimulation

MDCK cells were supplied by ATCC (Manassas, VA) and maintained in DMEM/F12
media with 15 mM HEPES and 10% FBS. Transfections were performed using
LipofectAMINE 2000 Reagent according to the manufacturer's protocol (Life
Technologies). Protein expression was confirmed by immunoblotting cell lysates
with antibodies directed to the specific tag used (HA, HAF7; c-myc, 9E10 or
A14) obtained from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA). Where
indicated, cells were incubated with wortmanin (Calbiochem, La Jolla, CA) (50
nM) in fresh media for 1 hour prior to activation. For HGF stimulation,
transfected cells were incubated overnight in serum-free media containing 0.2%
BSA and stimulated in the presence or absence of wortmanin for 10 minutes with
50 ng/ml HGF (R&D Systems, Minneapolis, MN). For Rac1 experiments, active
Rac1 (RacV12) or dominant-negative Rac1 (RacN17) was cotransfected with SGK1
and left overnight in media with 0.5% FBS prior to the kinase assay. For cAMP
activation, transfected cells were incubated overnight in 0.5% FBS and the
following morning treated with 1 mM 8Bromo-cAMP for 15 minutes before
harvesting. For adhesion-mediated activation, cells were serum-starved
overnight, trypsinized, washed twice and allowed to recover for 1 hour at
37°C in media with 0.2% BSA with or without wortmanin (50 nM) before
plating on fibronectin-coated dishes for the times indicated. Cell extracts
were prepared as previously described
(
Herrera, 1998;
Shelly et al., 1998) and
analyzed for protein expression and kinase activity.

Immunofluorescence

MDCK cells were plated on two chamber slides (Lab-Tek, Nalgene NUNC
International) and transfected with cDNA expression vectors encoding for
GFP-SGK1, the PH-domain of Akt (Akt/PH-GFP) or pEGFP-C1 (empty vector) with or
without vector encoding for Rac1 V12. After overnight incubation in 0.5% FBS,
the media was replaced and cells were stimulated with HGF (50 ng/ml) for 10
minutes or as indicated in the legend to the figure. The cells were washed and
fixed for 20 minutes in 4% formaldehyde then washed three times before
mounting using Aqua Poly/Mount (Polysciences, Inc., Warrington, PA). Confocal
Microscopy was performed on an Olympus Confocal microscope model IX70.

Adhesion-dependent modulation of FKHRL1 phosphorylation

MDCK cells transfected with either activated SGK1 (S/D) pKH3 or pKH3 (empty
vector) were serum starved overnight. The following morning, the cells were
trypsinized, washed and resuspended in media containing 0.2% BSA and 3 mM
EGTA. Cells were kept in suspension for 1 hour at 37°C (t=1 hour) or lysed
immediately (t=0). Total cell extracts were prepared as described in the
kinase assay section and analyzed for the presence of phosphorylated FKHRL1 by
western analysis. Anti-phospho-FKHRL1 (T32), (Cell Signaling Technologies) and
anti-FKHRL1 (Upstate Biotechnologies) antibodies were used.

Results

Hepatocyte growth factor (HGF) activates SGK1 in MDCK cells

Treatment of epithelial cells, such as MDCK, with HGF leads to increased
cell motility and biochemical changes that result in the scattering response
(
Stoker et al., 1987;
Weidner et al., 1990;
Weidner et al., 1993). Since
the scatter response of the cells to HGF requires PI-3K activity
(
Khwaja et al., 1998;
Potempa and Ridley, 1998), we
have analyzed whether treatment of MDCK cells with HGF would lead to
regulation of SGK activity in a manner similar to the activation of AKT by HGF
(
Coulonval et al., 2000;
Liu, 1999;
Xiao et al., 2001). We used
HA-tagged SGK1 to transiently transfect MDCK cells and measured
immunoprecipitated SGK1 activity in response to HGF treatment. In addition, we
have analyzed the site-specific phosphorylation of endogenous AKT to compare
its activation profile with the profile of SGK. As shown in
Fig. 1A, stimulation of the
cells with HGF (50 ng/ml) leads to a time-dependent activation of SGK1,
reaching a maximum at 10 minutes. We compared the fold of activation of
transfected SGK1 obtained by stimulation of MDCK cells with either that for
HGF or IGF-1. Under our assay conditions, MDCK cells stimulated with HGF
yielded activation of SGK1 to a similar extent as MDCK cells stimulated with
IGF-1 (data not shown). The activation profile of HA-SGK1 in response to HGF
parallels the phosphorylation of endogenous AKT at S473
(
Fig. 1B). This phosphorylation
has been previously shown to correlate with AKT activation
(
Vanhaesebroeck and Alessi,
2000). The HGF-induced activation of SGK1 or AKT is dependent on
the activity of PI-3K since two unrelated chemical inhibitors of PI-3K
activity, wortmanin and LY294002, prevent both AKT phosphorylation and SGK1
activation (
Fig. 1C). (For
LY294002 the data is not shown.) The activation of SGK1 by HGF was dependent
on the integrity of the phosphorylation sites present at T256 and S422 of
SGK1. Both single point mutants and the double site mutant SGK1 proteins
failed to respond to HGF stimulation (data not shown). A similar behavior has
been observed for IGF-1/H2O2 stimulation of SGK activity
(
Kobayashi and Cohen, 1999;
Park et al., 1999). We studied
the behavior of several mutant forms of SGK in response to various stimuli.
The substitution S422D (S/D) residue has been shown to produce an activated
form of SGK (
Kobayashi and Cohen,
1999;
Park et al.,
1999). In our MDCK assay system, the S422D mutant produces a
2.4±0.4 (n=7) fold greater activation than wildtype. Although
the SGK (S/D) has basal activity greater than wildtype, it still can be
moderately stimulated by an agonist (
Table
1).

Activation of SGK1 by HGF is wortmanin dependent. (A) Time course of SGK1
activation by HGF. MDCK cells were transiently transfected with HA-SGK1,
stimulated with HGF and at the indicated times, cells were harvested and
HA-SGK1 activity was measured as described in the Materials and Methods. The
lower insert depicts the amount of HA-SGK1 present in the assay at the
indicated times. (B) Time course activation of AKT by HGF. Cell extracts
prepared as in (A) were separated in a SDS-PAGE and analyzed for the presence
of phosphorylated AKT (T473)(pAKT) or total AKT as described in the Materials
and Methods. (C) HGF activation of HA-SGK1 or AKT is blocked by wortmanin.
MDCK cells were transfected with HA-SGK1, stimulated with HGF for 10 minutes
in the presence or absence (DMSO) of wortmanin, and the kinase activity
associated with HA-SGK1 was measured as in (A). The lower insert depicts the
amount of phosphorylated AKT (T473)(pAKT), total AKT and HA-SGK1 present under
the conditions described. Despite HGF-treated extract being under-loaded in
the AKT gel, it contains detectable levels of pAKT.

Rac1-V12 activates HA-SGK1

The stimulation of MDCK cells with HGF induces activation of the
Ras/Rac/Rho family of small GTP-binding proteins
(
Royal et al., 2000). We
investigated whether cotransfection of activated forms of Rac1, RhoA or Rap1
would lead to HA-SGK1 activation. As shown in
Fig. 2A, cotransfection of
activated Rac1 (Rac1 V12) with HA-SGK1, but not with activated RhoA or Rap1,
resulted in a highly reproducible activation of HA-SGK activity in a
dose-dependent manner (
Fig.
2B). Interestingly, the activation observed in response to
Rac1-V12 was not blocked by wortmanin (
Fig.
2C) although it still required an intact PDK1 (T256) or PDK2
(S422) phosphorylation site since activation was not observed in a SGK1
protein lacking either or both of these sites (data not shown). We next tested
whether the Rac1 pathway was involved in mediating the HGF activation of SGK1.
We compared the degree of HGF-induced activation of HA-SGK1 upon
contransfection with the dominant-negative form of Rac1 (RacN17)
(
Hall, 1998;
Symons and Settleman, 2000).
As shown in
Fig. 2D,
contransfection of HA-SGK1 with RacN17 did not prevent the activation of
HA-SGK by HGF. On the other hand, cotransfection of activated Rac1 with
HA-SGK1 leads to increased activation upon stimulation with HGF as compared
with the activity obtained in the presence of activated Rac1 alone or HGF
stimulation in the absence of activated Rac1 (data not shown).

Activation of HA-SGK1 by Rac1V12 is wortmanin independent. (A) MDCK cells
were co-transfected with HA-SGK1 and either Rac1V12 (lane 1), RhoL63 (lane 2),
Rap1V12 (lane3) or HA-SGK alone (lane 4), and the kinase activity associated
with HA-SGK1 was assayed as described in the Materials and Methods. The
results are presented as the fold increase in activation over the activity
obtained in the absence of the small GTP-binding proteins. The lower insert
depicts the amount of HA-SGK1 present in the assay. (B) Dose-dependent
activation of HA-SGK1 by Rac1V12. MDCK cells were cotransfected with HA-SGK1,
and the indicated amounts of Rac1V12 and the kinase activity was assayed as
described in the Materials and Methods. The lower insert depicts the amount of
HA-SGK1 present in the assay. (C) The effects of wortmanin on RacV12
activation of HA-SGK1. HA-SGK1 was cotransfected with Rac1V12, and cell
extracts were prepared from wortmanin or vehicle-treated cells as described in
the legend to
Fig. 1C. The
lower insert depicts the amount of HA-SGK1 present in the assay. (D) The
HGF-mediated activation of HA-SGK1 is not blocked by RacN17. MDCK cells were
cotransfected with HA-SGK and an empty vector or a vector containing
myc-tagged RacN17. The amount of SGK in the kinase assay is depicted in the
HA-SGK blot. Verification of myc-RacN17 expression is shown in the lower blot.
The kinase activity was carried out as described in the legend to
Fig. 2A.

Analysis of the primary structure of SGK1 reveals the presence of other
potential phosphorylation sites such as a tyrosine kinase site (Y124) and a
PKA site (T369). We analyzed whether activation of SGK1 results in tyrosine
phosphorylation and whether the potential PKA site is a functional regulatory
site. We failed to detect tyrosine phosphorylation of HA-SGK1 in response to
HGF treatment. Similarly, we were unable to demonstrate a significant
activation of HA-SGK1 by cAMP stimulation under conditions where we detect
cAMP-mediated phosphorylation of the transcription factor CREB
(
Fig. 3). In addition, we
mutated the potential PKA site and tested its role in the activation of
HA-SGK1 in response to stimulation. Changing the potential PKA phosphorylation
site to alanine produced an active protein that was still responsive to HGF or
Rac1 V12. The level of activation of T369A mutant enzyme by Rac1 V12 or HGF
was consistently only 75% of that the wild-type enzyme. While this work was
being carried out, a report was published
(
Perrotti et al., 2001)
describing cAMP regulation of SGK activity with results that are different
from those that we describe above. The reason for these discrepancies is
currently not clear.

SGK is not regulated by cAMP. Top, MDCK cells were transfected with
wild-type, Thr369A mutant or S422D mutant SGK and cells were stimulated with 1
mM cAMP analog before SGK1 activity was measured. Kinase assay was carried out
as described in the Materials and Methods. Bottom. The cell extracts were
probed for phosphorylated CREB to verify cAMP stimulation and probed with
anti-HA to determine the amount of HA-SGK in the kinase assay.

We constructed a GFP-SGK1 fusion protein in order to study whether
activation of SGK1 by HGF or RacV12 correlates with subcellular translocation.
We compared GFP-SGK1 movement with that of the GFP-PH/AKT fusion protein,
which is a marker for membrane recruitment
(
Raucher et al., 2000). As
shown in
Fig. 4A, the GFP-SGK1
fusion protein is expressed as a full-length, kinase-active protein in
transfected MDCK cells. Treatment of GFP-SGK1- or GFP-PH/AKT-transfected cells
with either HGF or cotransfection with RacV12 induces recruitment of the
GFP-PH/AKT protein to the intercellular surfaces
(
Fig. 4B), indicating that
activation of PI-3K has taken place. The GFP-SGK1 protein, by contrast, is not
significantly recruited to the same structures, suggesting that HGF-and
RacV12-mediated activation of SGK1 does not involve significant subcellular
redistribution.

SGK activation does not correlate with membrane translocation. (A) GFP-SGK1
is a kinase-active protein. MDCK cells were transfected with empty vector
(GFP) or GFP-SGK1 (SGKwt) and stimulated by HGF for 15 minutes. Kinase
activity was determined as described in the Materials and Methods. The lower
insert depicts the amount of GFP-SGK present in the assay. (B) MDCK cells were
transfected with GFP, GFP-SGK or GFP-PH/AKT (the pleckstrin homology domain of
AKT) and stimulated with HGF for 15 minutes. A parallel cotransfection
experiment between the above vectors and RacV12 is also shown. After fixing
the cells, the location of the GFP derivatives was determined by confocal
microscopy as described in the Materials and Methods.

Adhesion-dependent activation of SGK1

It has previously been demonstrated that cell adhesion to the extracellular
matrix via integrins induces activation of AKT
(
Banfic et al., 1998;
Guilherme and Czech, 1998).
Thus, we were interested in determining whether adhesion of MDCK cells to
fibronectin would result in activation of transfected HA-SGK1. We measured and
compared the kinase activity of SGK1 isolated from suspended and attached
cells. We also followed the endogenous activation of AKT by monitoring its
phosphorylation at S473.

Time course analysis of the activation of SGK1 upon interaction of MDCK
cells with immobilized fibronectin showed that adhesion of the cells to the
extracellular matrix induces a prolonged activation of SGK1
(
Fig. 5A). The activation
profile followed the spreading of the cells (data not shown). As previously
reported (
King et al., 1997),
the adhesion of the cells also leads to AKT activation
(
Fig. 5B). However, it appears
that the integrin-mediated activation of HA-SGK1 differs from AKT activation
in that AKT phosphorylation was blocked by wortmanin (or LY294002). Although
the basal activity of HA-SGK1 in the presence of wortmanin is reduced, the
fold of activation was not (
Fig.
5C,D). Similarly, LY294002 did not reduce the fold activation of
SGK1 despite strong inhibition of AKT phosphorylation (data not shown).

Adhesion of MDCK cells to fibronectin activates HA-SGK1 in a
wortmanin-insensitive manner. (A) Time course of HA-SGK1 activation upon cell
adhesion to fibronectin-coated plates. Transfected MDCK cells were allowed to
bind to fibronectin-coated plates, and HA-SGK1 activity was measured at the
indicated times as described in the Materials and Methods. The insert depicts
the level of HA-SGK1 present in the assay. (B) Adhesion-dependent activation
of AKT. Cell extracts prepared as in (A) and were analyzed for the presence of
phosphorylated AKT (pAKT). (C) Adhesion-mediated activation of HA-SGK1 is not
blocked by wortmanin. HA-SGK1 activation by adhesion to fibronectin coated
plates was studied in the presence or absence of wortmanin. After 30 minutes
of adhesion to fibronectin, SGK1 activity was measured as in (A). The insert
depicts the level of HA-SGK1 present in the assay. (D) Adhesion-dependent
activation of AKT is blocked by wortmanin. Cells extracts were prepared as in
(C) and were analyzed for the presence of phosphorylated AKT (T473)
(pAKT).

Integrin-mediated activation of HA-SGK1 requires the intact phosphorylation
sites that regulate HA-SGK1 activation in response to HGF or Rac 1-V12. We
therefore assessed the role of Rac1 in integrin-mediated activation of SGK1.
Cotransfection of HA-SGK with dominant-negative Rac1 (RacN17) did not result
in inhibition of adhesion-stimulated activation of SGK1
(
Fig. 6). The RacN17 is acting
as a true dominant negative in this system since it blocked the HGF-stimulated
actin reorganization (data not shown). Cotransfection of HA-SGK1 with RacV12
results in increased activity in adherent cells compared with the effect of
transfection of HA-SGK1 alone. The binding of cotransfected cells to
fibronectin resulted in a greater activation than is obtained in the absence
of RacV12, indicating that the adhesion-dependent activation is achieved by a
parallel Rac-independent pathway (
Fig.
6).

Adhesion-mediated activation of HA-SGK1 is not blocked by Rac1N17. MDCK
cells were cotransfected with HA-SGK1 and myc-Rac1V12 or myc-Rac1N17, allowed
to adhere to fibronectin and the activation of HA-SGK1 was measured as
described in
Fig. 1. The lower
insert depicts the levels of HA-SGK1, Rac1N17 and Rac1V12 present in the
assay.

The activation of SGK1 by integrin or HGF may be connected to the
modulation of cell survival. HGF activates an anti-apoptotic pathway
(
Bardelli et al., 1996) and
blocks apoptosis of MDCK cells induced by loss of integrin-mediated cell
attachment (anoikis) (
Frisch and Francis,
1994), suggesting that both HGF and integrin signaling share a
common pathway. We investigated whether the phosphorylation state of FKHRL1, a
known modulator of cell survival (
Liu et
al., 2000), was regulated by cell attachment and whether this
regulation could be influenced by SGK1. As shown in
Fig. 7, deattachment of MDCK
cells leads to a rapid loss of phosphorylated FKHRL1 (compare lanes 1-4 with
lanes 7,8).

However, the presence of activated SGK1 prevented the dephosphorylation of
FKHRL1 during the incubation period, suggesting that activated SGK1 provides
survival signals similar to the one described for Akt
(
Brunet et al., 1999).

Discussion

SGK gene expression is under the control of growth factors and
glucocorticoids (
Webster et al.,
1993a;
Webster et al.,
1993b). Northen blot analysis has shown that this kinase is
expressed in several tissues, including the pancreas, skeletal muscle, liver,
heart, placenta, kidney and brain
(
Waldegger et al., 1997). The
expression of SGK has been correlated with aldosterone-mediated regulation of
the epithelial sodium channel (
Alvarez de
la Rosa et al., 1999;
Chen et
al., 1999). In addition, the activity of the heterodimeric
amino-acid transporter 4F2hc/LAT1 is associated with a non-selective cation
channel that is regulated by SGK1 (
Wagner
et al., 2000). The expression of SGK1 is regulated by anisotonic
and isotonic alterations of cell volume
(
Waldegger et al., 1997) as
well as by hyperosmotic stress (
Bell et
al., 2000). It has been shown that the expression of SGK1 is
deranged in diabetic nephropathy (
Lang et
al., 2000).

The role of SGK in signal transduction is poorly defined. However, as it
belongs to the AGC class of kinases, its activity is regulated by PDK1,
suggesting that it may participate in some of the pathways under the control
of PI-3K (
Currie et al., 1999;
Czech, 2000;
Kobayashi and Cohen, 1999;
Park et al., 1999;
Vanhaesebroeck and Alessi,
2000;
Williams et al.,
2000). Accordingly, it has been suggested that SGK participates in
the regulation of GSK-3 (
Kobayashi and
Cohen, 1999) and that it functionally replaces the Ypk1
gene in budding yeast (
Casamayor et al.,
1999). On the other hand, it is also evident from the data
presented in this paper that SGK1 activation can be accomplished by signaling
pathways that are independent of PI-3K activation. While this paper was in
preparation, a report describing activation of SGK by ERK5 independently of
PDK1-mediated activation was published (Hayashi, 2001).

We analyzed the activation profiles of AKT and SGK upon stimulation with
agonists or signaling intermediaries in order to compare the mechanism of
activation of these two members of the AGC kinase family. As described in the
Results section, we demonstrated that treatment of MDCK cells with HGF induces
activation of both AKT and SGK in a PI-3K-dependent manner
(
Fig. 1A-C;
Table 1). However, in contrast
to HGF-mediated activation of SGK1, activation of SGK by RacV12 was shown to
be wortmanin insensitive, suggesting that RacV12 activation does not require
activation of PI-3K (
Fig.
2A-C). It has been shown that Rac1-mediated activation of AKT in T
cells requires PI-3K activity (
Genot et
al., 2000). It remains to be determined whether Rac1 activates AKT
in MDCK cells and whether it is PI-3K independent as has been reported for the
activation of AKT by cAMP (
Filippa et al.,
1999). We analyzed the possibility that cAMP also activates SGK,
as treatment of granulosa cells with forskolin induces SGK phosphorylation
(
Gonzalez-Robayna et al.,
2000). We observed the presence of a potential PKA site in SGK and
mutated it to measure its contribution to SGK activation. Unlike researchers
in previous reports (
Perrotti et al.,
2001), we failed to demonstrate cAMP-mediated activation of SGK
under conditions that lead to phosphorylation of CREB
(
Fig. 3).

Our analysis of adhesion-dependent activation of SGK revealed that adhesion
of MDCK cells to fibronectin activates SGK in a wortmanin-independent manner.
This is in contrast to observations for AKT activation under the same
conditions (
Fig. 5A-C).
Integrin-mediated activation of SGK was not inhibited by the neutralization of
Rac activation. Indeed, we saw an additive activation mediated by RacV12 and
fibronectin adhesion, indicating that there are other pathways that lead to
SGK activation. Significantly, the activation of SGK is still under the
structural requirements that regulate PDK1 activation of SGK, since mutations
in the PDK1 or PDK2 sites produce an inactive SGK protein. There are
conditions, however, in which PDK1 activity does not require input from the
PI-3K pathway (
Vanhaesebroeck and Alessi,
2000), thus explaining the need for the PDK1 site without
requiring activation of PI-3K.

The regulation of SGK activity in MDCK cells by both HGF and cell adhesion
suggests that SGK may participate in signaling pathways that modulate cell
motility. However, recently published information
(
Brunet et al., 2001;
Liu et al., 2000) points to a
functional role for SGK that is complementary to the role that AKT plays in
promoting cell survival by directly phosphorylating and inactivating the
proapoptotic proteins FKHR and BAD (
Brunet
et al., 1999). Here we have shown that SGK1 also mediates
signaling associated with cell survival. The activation of SGK1 by HGF or
integrin may connect this protein to the protection of MDCK from anoikis since
the deattachment-induced dephosphorylation of FKHRL1 is significantly reduced
by expression of activated SGK1 (
Fig.
7).

In conclusion, we present experimental evidence that SGK1 activation can be
accomplished via HGF, PI-3K-dependent pathways and by integrin-mediated,
PI-3K-independent pathways. In addition, activation of SGK1 by the small
GTP-binding protein Rac1 has been observed. These results suggest that
activation of SGK1 could be achieved through signaling pathways involved in
the regulation of cell survival, cell-cell and cell-matrix interactions.

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